Diacylglycerol kinase alpha is a proliferation marker of intrahepatic cholangiocarcinoma associated with the prognosis

Abstract Background Intrahepatic cholangiocarcinoma (ICC) has a high recurrence rate and a poor prognosis. Thus, the development of effective treatment and prognostic biomarkers is required. High expression of diacylglycerol kinase alpha (DGKα) is a prognostic factor for the recurrence of hepatocellular carcinoma. However, the relationship between DGKα expression and prognosis in ICC has not been reported. Methods Immunohistochemistry (IHC) with anti‐DGKα antibody was performed on surgical specimens of ICC (n = 69). First, DGKα expression in cancer cells was qualitatively classified into four groups (−, 1+, 2+, 3+) and divided into two groups (DGKα− and DGKα+1 + to 3+). The relationship between clinical features and DGKα expression was analyzed. Second, Ki‐67 expression was evaluated as a cell proliferation marker. The number of Ki‐67‐positive cells was counted, and the relationship with DGKα expression was examined. Results DGKα IHC divided the patients into a DGKα+ group (1+: n = 15; 2+: n = 5; 3+: n = 5) and a DGKα− group (−: n = 44). In the DGKα+ group, patients were older and had advanced disease. Both overall survival and recurrence‐free survival (RFS) were significantly worse in the DGKα+ patients. DGKα+ was identified as an independent prognostic factor for RFS by multivariate analysis. Furthermore, the number of Ki‐67‐positive cells increased in association with the staining levels of DGKα. Conclusion Pathological DGKα expression in ICC was a cancer proliferation marker associated with recurrence. This suggests that DGKα may be a potential therapeutic target for ICC.

Methods: Immunohistochemistry (IHC) with anti-DGKα antibody was performed on surgical specimens of ICC (n = 69).First, DGKα expression in cancer cells was qualitatively classified into four groups (−, 1+, 2+, 3+) and divided into two groups (DGKα− and DGKα+1 + to 3+).The relationship between clinical features and DGKα expression was analyzed.Second, Ki-67 expression was evaluated as a cell proliferation marker.The number of Ki-67-positive cells was counted, and the relationship with DGKα expression was examined.
Furthermore, the number of Ki-67-positive cells increased in association with the staining levels of DGKα.

Conclusion:
Pathological DGKα expression in ICC was a cancer proliferation marker associated with recurrence.This suggests that DGKα may be a potential therapeutic target for ICC.

| INTRODUCTION
Primary liver cancer is one of the most intractable gastrointestinal tumors and most cases are hepatocellular carcinoma (HCC), but the next most common histological type is intrahepatic cholangiocarcinoma (ICC).ICC is a type of cholangiocarcinoma that arises peripherally from the secondary branches.3][4] The incidence is increasing worldwide, including in Japan, 1 but the cause has not been identified.Although surgery is the first-line curative treatment, the resection rate at diagnosis is less than 60% 5,6 and the postoperative recurrence rate is more than 70%, [7][8][9] and no effective nonsurgical treatment such as drug therapy has yet been established.Against this background, there is an urgent need to identify effective treatments and biomarkers to improve the poor prognosis of this disease.
2][13] Inhibition of DGKα induces apoptosis in DGKαexpressed cancer cell lines regardless of their origins. 12So, DGKα has been thought of as an important cell growth factor in cancers.
On the other hand, DGKα has been reported as a key molecule inducing the anergy of T cells. 14Using a liver tumor model, it has been shown that DGKα inhibitors exert their antitumor effects via T cell immunostimulant, 11 and basic studies have shown that inhibitors better inhibit cell proliferation in multiple cancer cell lines with DGKα expression. 12DGKα inhibition is considered a novel cancer therapeutic target. 15egarding the evaluation of immunostaining in human cancer tissues, there is a report related to the prognosis of colorectal cancer (CRC) patients.CRC stage II (pT3N0M0) patients who highly expressed DGKα in cancer cells and stromal cells that were considered equal to tumor-infiltrating lymphocytes had a worse prognosis. 13n this study, we investigated the relationship between DGKα expression and the clinicopathological features and prognosis of ICC.

| Patient selection and clinical treatment
Seventy ICC patients underwent hepatectomy at Hokkaido University Hospital from 1997 to 2013.Among these, the histopathology and clinical outcomes of 69 patients were analyzed; 1 case was excluded due to a lack of appropriate pathological specimens.Patients who were diagnosed with ICC preoperatively underwent hepatectomy and lymph node dissection of the hepatoduodenal ligament and the area around the common hepatic artery and retro pancreas head.When the tumor was located in the left lobe, the lymph nodes around the lesser curvature of the stomach were also dissected.When the tumor had bile duct invasion, the extrahepatic bile duct was resected and biliary reconstruction was performed.Patients from 2000 to 2007 were treated with 5-fluorouracil-based adjuvant chemotherapy, and patients after 2007 were treated gemcitabine-based adjuvant chemotherapy.Patients were followed up every 3 months until 5 years after surgery.The maximum follow-up was 13 years, and there were 5 patients whose hospital visits were discontinued within 5 years for reasons other than death.

| Immunohistochemical staining
An original anti-DGKα monoclonal antibody named DaMab-8 was produced by Sano et al. 16 and donated from our collaborator, Ono Pharmaceutical Co., Ltd.Four-micrometer-thick sections of formalin-fixed, paraffin-embedded specimens were used for immunohistochemical staining.After deparaffinization, antigen retrieval was performed using a citric acid buffer and heated for 20 min at 95°C, and endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide at room temperature for 10 min.Sections were washed with Tris-buffered saline and then incubated with antihuman DGKα monoclonal antibodies (DaMab-8, 1:500) overnight at 4°C, or with anti-Ki-67 monoclonal antibodies (ab16667, Abcam), followed by incubation at room temperature for 30 min with Histofine Simple Stain, MAX PO (MULTI).Proteins were visualized biomarker, diacylglycerol kinase, intrahepatic cholangiocarcinoma, primary liver cancer, prognostic factor using 3-3′-diaminobenzidine-4HCL at room temperature for 5 min, followed by counterstaining with Mayer's hematoxylin.

| Evaluation
DGKα staining in cancer cells was evaluated at four levels according to the intensity of staining.Patients were classified as stain positive when the cytoplasm of cancer cells was recognized as positively stained in the high-power field of view and were classified into four levels according to their staining intensity.In all samples, there were peritumoral inflammatory cells like lymphocytes that were positive for staining, and the staining level was confirmed to be the same.Ki-67 staining was calculated as the percentage of positively stained cells out of the total number of cancer cells in each of the four intensely magnified fields.Cell counts were performed using ImageJ version: 2.1.0/1.53c.At least 500 cancer cells per patient were included in the field of view.The scores were evaluated by two different medical doctors in a double-blind manner, and the lower scores were used if the score differed depending on the evaluator.

| Statistical analysis
Comparisons of the relationship between background factors and DGKα were made using Fisher's exact test for categorical variables and the Mann-Whitney.
U test for continuous variables.Survival curves were estimated using the Kaplan-Meier method, and the differences in survival rates between groups were compared by the log-rank test.Univariate and multivariate analyses were performed using Cox's proportional hazards regression model to evaluate independent factors predictive of patient survival.Multivariate analysis was performed using the factors extracted in the univariate analysis.In analyses related to recurrence-free survival (RFS), cases with curability C were excluded.
When DGKα was classified into positive and negative groups, the Mann-Whitney U test was used for comparison with the Ki-67-positive cells.However, when DGKα was classified according to staining intensity, the Kruskal-Wallis test was used followed by multiple comparisons using the Steel-Dwass test.p-Values of <0.05 were considered to be significant.
All statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University,

| Evaluation method of DGKα immunohistochemistry in cases of ICC
Patients' backgrounds are shown in Table 1.DGKα staining of cancer tissues from the patients was classified into three levels according to the staining intensity in the cancer cell cytoplasm, and cases in which cancer cells in the sections could not be judged to be specifically stained were defined as negative for staining.Representative images of immunohistochemical staining for DGKα are shown in Figure 1.Forty-four cases were negative (−), 15 cases were weakly positive (1+), 5 cases were moderately positive (2+), and 5 cases were strongly positive (3+).

DGKα-positive cases
In the DGKα-positive group (1+ to 3+), patients were older and had higher CA19-9 levels and more advanced disease.There were no differences in tumor size, number of tumors, tumor differentiation, lymph node metastasis, degree of vascular invasion, and surgical factors (resection volume and curability) between the DGKα-positive and -negative groups (Table 2).

OS and RFS
Survival time analysis was performed and Kaplan-Meier curves are shown in Figure 2. In the log-rank test, OS at 5 years after surgery was p = 0.0423 (Figure 2A) and RFS was p = 0.00857 (Figure 2B).In both cases, the DGKαpositive group had a significantly poor prognosis.

| DGKα positivity is an independent poor prognostic factor for RFS in multivariate analysis
Regression analysis of each clinicopathological factor showed that, for OS (Table 3), the prognostic factors identified in the univariate analysis were sex, number of tumors, lymph node metastasis, vascular invasion, pathological stage, and surgical curability.In the multivariate analysis, sex, number of tumors, lymph node metastasis, vascular invasion, and surgical radiosurgery were independent prognostic factors.For RFS ( prognostic factors in the univariate analysis.In the multivariate analysis, tumor number and DGKα positivity were independent prognostic factors.

| DGKα expression intensity correlates with the number of Ki-67positive cells
Ki-67 staining was performed using sections of tissue taken near the tissue used for DGKα staining.A representative image is shown in Figure 3A.The number of positively stained cells was evaluated, and the number of Ki-67-positive cells was predominantly increased in the DGKα-positive group (Figure 3B,D), while the number of Ki-67-positive cells was higher in the group with higher DGKα intensity (Figure 3C,E).Because Ki-67 staining positivity in cancer cells is known to be a proliferation marker, increased DGKα expression in ICC may contribute to the proliferation of ICC.

| DISCUSSION
In patients with ICC in this study, those with positive DGKα expression were older and at an advanced pathological stage, and had higher CA19-9 levels.They had a significantly poorer prognosis regarding both OS and RFS.In the multivariate analysis of prognostic factors in RFS, high DGKα expression was an independent poor prognostic factor.Furthermore, DGKα expression intensity was correlated with the positive rate of Ki-67 cells and was found to be a factor associated with cancer cell proliferation.
It has been reported that DGKα inhibition induces cancer-cell apoptosis. 11,12,17A DGKα-specific inhibitor was more effective in inhibiting proliferation in the cell line, which had higher DGKα expression. 12Increased DGKα expression may increase the dependence of cancer growth on DGKα function, and pathological evaluation of tumor tissue can indicate the targets of DGKα inhibitory therapy.
It is expected to become a therapeutic target across various cancers from the previous reports.Nevertheless, it has never been investigated DGKα in ICC.This is the first report to investigate the relationship between the pathological DGKα expression level and the clinicopathological features in cholangiocarcinoma.
In addition, in the present study, Ki-67 staining was evaluated by the previously used evaluation system in HCC cases. 10DGKα expression can evaluate cell proliferation regardless of the histological type of primary liver cancer.We have confirmed the antitumor effects of DGKα inhibitors using a mouse model of liver cancer. 11These results from human clinical specimens, which revealed similar features in HCC and ICC, provide evidence to optimize the therapeutic indications for DGKα inhibitor therapy in almost all primary liver cancers.
The chemotherapy of biliary tract cancers has advanced rapidly in the past decade.Immune checkpoint inhibitors have also become part of standard therapy, 18 such as durvalumab and pembrolizumab which inhibit the PD-1/PD-L1 pathway.DGKα was reported as a regulator of T cells and DGKα inhibitor is expected as a target of cancer immunotherapy.Our previous research revealed that the antitumor effect of DGKα inhibitor may be synergistically enhanced when used in combination with a PD-L1 antibody. 11n this study, DGKα expression was not an independent factor of OS in the multivariate analysis.This may be because some patients who got noncurative resection received advanced treatments such as chemotherapy or local therapy.Therefore, we consider that the difference was extracted as an independent prognostic factor only concerning RFS.
A limitation of this study is that the data are from a limited number of patients at a single institution.We have not yet found any evidence of DGK expression or activity by other means than tissue staining biomarkers.
We believe that DGKα is a promising biomarker and therapeutic target molecule for primary liver cancer, including not only HCC but also ICC.We hope that the DGK-related molecular mechanisms will be elucidated and that along with the development of DGKα inhibitory therapy.

T A B L E 1
Abbreviation: ICC, Intrahepatic cholangiocarcinoma.
Univariate and multivariate analyses of prognostic factors related to overall survival.Univariate and multivariate analyses of prognostic factors related to recurrence-free survival.
T A B L E 3Abbreviations: CI, confidence interval; DGKα, diacylglycerol kinase alpha; HR, hazard ratio; OS, overall survival.T A B L E 4